The goal of this grant has been to develop an approach to inducing mixed hematopoietic chimerism across MHC barriers with sufficiently high reliability and low toxicity that it could be used for the induction of organ allograft tolerance in humans. In the currently funded grant, we have utilized costimulatory blockade to reliably tolerize pre-existing peripheral and intrathymic CD4 and CD8 T cells to allogeneic marrow donors, achieving mixed chimerism and eventual central, deletional tolerance in the maintenance phase. Pre-existing peripheral and intrathymic mature alloreactive T cells are tolerized by allogeneic BMT with one injection of anti-CD154 mAb. Peripheral CD8 cells are more reliably tolerized if a low dose of TBI (3 Gy) is given 1 day prior to BMT. Peripheral CD4 cell tolerance involves anergy followed by deletion due to antigen presentation on APC lacking an activation signal through CD40. Donor-reactive peripheral CD8 cells undergo even more rapid deletion than CD4 cells, and only CD8 tolerance, remarkably, requires recipient B cells and indirect allorecognition by recipient CD4 cells. We have obtained evidence for a novel indirect CD8 cell-mediated rejection pathway of cellular allograft rejection, which must be tolerized in our model. We now propose: 1) To identify molecular pathways through which BMT with anti-CD154 leads to tolerization of peripheral donor- reactive CD4 T cells. The ultimate goal of this aim is to identify reagents that could replace anti-CD154 by targeting pathways downstream of the blocked CD40/CD154 interaction; 2)To identify the CD4/CD8/APC interactions leading to peripheral CD8 cell tolerance when 3 Gy TBI on Day -1 and anti-CD154 are given. We hypothesize that separate interactions between directly alloreactive CD4 and CD8 cells and indirectly alloreactive CD4 and CD8 cells and donor and host APC, respectively, tolerize two separate pathways of CD8-mediated marrow rejection; 3)To identify the mechanisms by which recipient B cells promote tolerance of alloreactive CD8 cells. We hypothesize that Ig receptors on recipient B cells recognizing donor alloantigens permit the concentrated presentation of donor MHC-derived peptides on both class I and class II molecules of the B cell, which mediates tolerance of indirectly alloreactive CD4 and CD8 cells. Achievement of these goals will lead to a greater understanding of the pathways to allogeneic marrow rejection and tolerance and will allow the identification of molecular pathways downstream of CD40 blockade to target in the development of new strategies for achieving this robust tolerance. These studies will ultimately enhance our ability to reliably achieve marrow engraftment without toxicity in patients needing organ transplants and those with hematologic disorders such as hemoglobinopathies. [unreadable] [unreadable] [unreadable]